The proposed studies will focus primarily on supramolecular aspects of biological membranes. Using a combination of freeze-etching, freeze-substitution, negative staining, SDS-polyacrylamide gel electrophoresis and immunolabeling techniques, we will investigate a) initial structural events of membrane fusion that occur within the millisecond range, together with related mechanism of membrane growth and turnover; b) membrane-microtubule (MT) interactions; c) structure-function correlations of chloroplast membrane components, coupled with changes in their architecture caused by light activation. Spray-freezing (Bachmann and Schmitt 1971), which permits freezing at a rate of greater than negative 10,000 degrees C/sec, will be of particular importance for obtaining the desired time resolution in the studies of membrane fusion and of light activation of chloroplast membranes. The sequence of events associated with membrane fusion will first be analyzed in water-secreting systems of selected protozoa and algae, and later extended to protein-secreting animal cells and to tissue culture cells. A trypanosomatid flagellate, whose plasma membrane can be isolated with an attached cytoskeleton of MT's will serve as starting material for a structural and biochemical characterization of the linkage between MT's and membranes. Finally, the chloroplast studies will focus on refining the supramolecular model of chloroplast membranes we have already developed, by mapping the distribution of specific proteins using an improved immunolabel which we plan to develop.